Tuberization in potato is a complex developmental process involving the expression of a specific set of genes leading to the synthesis of tuber proteins. We here report the cloning and analysis of mRNAs encoding tuber proteins. From a potato tuber cDNA library four different recombinants were isolated which hybridized predominantly with tuber mRNAs. Northern blot hybridization experiments showed that three of them, pPATB2, p303 and p340, can be regarded as tuber-specific while the fourth, p322, hybridizes to tuber and stem mRNA. Hybrid-selected in vitro translation and nucleotide sequence analysis indicate that pPATB2 and p303 represent patatin and the proteinase inhibitor II mRNA respectively. Recombinant p322 represents an mRNA encoding a polypeptide having homology with the soybean Bowman-Birk proteinase inhibitor while p340 represents an mRNA encoding a polypeptide showing homology with the winged bean Kunitz trypsin inhibitor. In total, these four polypeptides constitute approximately 50% of the soluble tuber protein. Using Southern blot analysis of potato DNA we estimate that these mRNAs are encoded by small multigene families.
Establishment of a nitrogen-fixing root nodule is accompanied by a developmentally regulated expression of nodulin genes, only some of which, the so-called early nodulin genes, are expressed in stages preceding actual nitrogen fixation. We have isolated soybean cDNA clones representing early nodulin genes and have studied clone pENOD2 in detail. The cDNA insert of this clone hybridizes to nodulespecific RNA of 1200 nucleotides in length. The RNA that was hybrid-selected by the cloned ENOD2 DNA was in vitro translated to produce two nodulins with an apparent Mr of 75,000, the N-75 nodulins. These two nodulins differ slightly in charge and one does not contain methionine. The amino acid sequence deduced from the DNA sequence shows that proline accounts for 45% of the 240 residues in these nodulins and the sequence contains at least 20 repeating heptapeptide units. The amino acid composition of none of the (hydroxy)proline-rich (glyco)proteins described in plants resembles the composition of the N-75 nodulins, especially with respect to the high glutamic acid and the low serine content. This suggests that the N-75 nodulins belong to a hitherto unidentified class of presumably structural proteins. The genes encoding the N-75 nodulins were found to be expressed in nodule-like structures devoid of intracellular bacteria and infection threads, indicating that these nodulins do not function in the infection process but more likely function in nodule morphogenesis.The formation of nitrogen-fixing nodules on the roots of leguminous plants induced by bacteria of the genera Rhizobium and Bradyrhizobium involves the specific expression of a number of plant genes called nodulin genes (1-3). In a description of nodule development, Vincent (4) distinguishes between three stages in nodule development denoted as "preinfection", "infection and nodule formation", and "nodule function". In the preinfection stage, the Rhizobium bacteria recognize their host plants and attach to the root hairs, an event that is followed by root hair curling. At the moment, nothing is known about specific plant genes that are involved in this stage. In the next stage, the bacteria enter the roots by infection threads while concomitantly the dedifferentiation of some cortical cells results in the formation of meristems. The infection threads grow toward the meristematic cells; bacteria are released into the cytoplasm of about half of these cells and develop into bacteroids. In the final stage, further differentiation of nodule cells occurs leading up to a nitrogen-fixing nodule. Most studies on the expression of nodulin genes so far have been confined to the final stage of root nodule development. But the steps involved in root nodule formation show that major decisions determining the development of a root nodule are made in the stages preceding the establishment of a nitrogen-fixing nodule. We have shown (5) that nodulin genes are differentially expressed during development and that in pea at least two nodulin genes are transcribed in the seco...
SummaryTo devise a method for function-based gene isolation and characterization in barley, we created a plasmid containing the maize Activator (Ac) transposase (AcTPase) gene and a negative selection gene, codA, and a plasmid containing Dissociation (Ds) inverted-repeat ends surrounding the selectable herbicide resistance gene, bar. These plasmids were used to stably transform barley (Hordeum vulgare). In vitro assays, utilizing a Ds-interrupted uidA reporter gene, were used to demonstrate high-frequency excisions of Ds when the uidA construct was introduced transiently into stably transformed, AcTPaseexpressing plant tissue. Crosses were made between stably transformed plants expressing functional transposase under the transcriptional control of either the putative AcTPase promoter or the promoter and ®rst intron from the maize ubiquitin (Ubi1) gene, and plants containing Ds-Ubi-bar. In F 1 plants from these crosses, low somatic and germinal transposition frequencies were observed; however, in F 2 progeny derived from individual selfed F 1 plants, up to 47% of the plants showed evidence of Ds transposition. Further analyses of F 3 plants showed that approximately 75% of the transposed Ds elements reinserted into linked locations and 25% into unlinked locations. Transposed Ds elements in plants lacking the AcTPase transposase gene could be reactivated by reintroducing the transposase gene through classical genetic crossing, making this system functional for targeted gene tagging and studies of gene function. During the analysis of F 3 plants we observed two mutant phenotypes in which the transposed Ds elements co-segregate with the new phenotype, suggesting the additional utility of such a system for tagging genes.
Site-specific integration is an attractive method for the improvement of current transformation technologies aimed at the production of stable transgenic plants. Here, we present a Cre-based targeting strategy in Arabidopsis (Arabidopsis thaliana) using recombinase-mediated cassette exchange (RMCE) of transferred DNA (T-DNA) delivered by Agrobacterium tumefaciens. The rationale for effective RMCE is the precise exchange of a genomic and a replacement cassette both flanked by two heterospecific lox sites that are incompatible with each other to prevent unwanted cassette deletion. We designed a strategy in which the coding region of a loxP/lox5171-flanked bialaphos resistance (bar) gene is exchanged for a loxP/lox5171-flanked T-DNA replacement cassette containing the neomycin phosphotransferase (nptII) coding region via loxP/loxP and lox5171/ lox5171 directed recombination. The bar gene is driven by the strong 35S promoter, which is located outside the target cassette. This placement ensures preferential selection of RMCE events and not random integration events by expression of nptII from this same promoter. Using root transformation, during which Cre was provided on a cotransformed T-DNA, 50 kanamycinresistant calli were selected. Forty-four percent contained a correctly exchanged cassette based on PCR analysis, indicating the stringency of the selection system. This was confirmed for the offspring of five analyzed events by Southern-blot analysis. In four of the five analyzed RMCE events, there were no additional T-DNA insertions or they easily segregated, resulting in highefficiency single-copy RMCE events. Our approach enables simple and efficient selection of targeting events using the advantages of Agrobacterium-mediated transformation.
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